1. Field of the Invention
The present invention relates to a herpes viral mutant capable of selectively targeting tumor cells and/or other specific cell populations. More particularly, the present invention relates to the use of cell-specific and/or tumor-specific promoters to retarget mutant herpes viral vectors toward tumors and specific cell types. The cell-specific and/or tumor-specific promoter is used to drive expression of the herpes gamma (γ) 34.5 gene, whose gene product is responsible for producing large quantities of progeny virus in infected cells.
Herpes vectors without the γ34.5 gene do not replicate well, which is desirable for clinical use. However, the absence of the γ34.5 gene, also diminishes the ability of the virus to kill tumors or any other infected tissue. The present invention allows for the production of high amounts of herpes virus in cells that can use the cell- and/or tumor-specific promoter. Cells that cannot turn on the promoter, however, do not support viral replication, thus saving them and their neighboring cells from active and noxious viral infection and replication, thus, redirecting herpes' virulence towards desired target cells.
2. Related Art
A. Conventional Cancer Therapies
Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites (metastases) it will likely result in death of the individual.
In 1999, in the United States alone, approximately 563,100 people, or about 1,500 people per day, are expected to die of cancer. (Landis, et al., “Cancer Statistics, 1999,” CA Canc. J. Clin. 49:8-31 (1999)). Moreover, cancer is a leading cause of death among children aged 1 to 14 years, second only to accidents. Id. Thus, clearly there is a need for the development of new cancer therapies.
1. Common Limitations of Conventional Therapies
The desired goal of cancer therapy is to kill cancer cells preferentially, without having a deleterious effect on normal cells. Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy, and chemotherapy.
Surgery was the first cancer treatment available, and still plays a major role in diagnosis, staging, and treatment of cancer, and may be the primary treatment for early cancers (see, Slapak, C. A. and Kufe, D. W., “Principles of Cancer Therapy,” in Harrison 's Principles of Internal Medicine, Fauci, A. S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, at 524). However, although surgery may be an effective way to cure tumors confined to a particular site, these tumors may not be curable by resection due to micrometastatic disease outside the tumor field. Id. Any cancer showing a level of metastasis effectively cannot be cured through surgery alone. Id.
Radiation therapy is another local (nonsystemic) form of treatment used for the control of localized cancers. Id at 525. Many normal cells have a higher capacity for intercellular repair than neoplastic cells, rendering them less sensitive to radiation damage. Radiation therapy relies on this difference between neoplastic and normal cells in susceptibility to damage by radiation, and the ability of normal organs to continue to function well if they are only segmentally damaged. Id. Thus, the success of radiation therapy depends upon the sensitivity of tissue surrounding the tumor to radiation therapy. Id. Radiation therapy is associated with side effects that depend in part upon the site of administration, and include fatigue, local skin reactions, nausea and vomiting. Id at 526. In addition, radiation therapy is mutagenic, carcinogenic and teratogenic, and may place the patient at risk of developing secondary tumors. Id.
Other types of local therapy have been explored, including local hyperthermia (Salcman, M., et at., J. Neuro-Oncol. 1:225-236 (1983)), photoradiation therapy (Cheng, M. K., et al., Surg. Neurol. 25:423-435 (1986)), and interstitial radiation (Gutin, P. H., et al., J. Neurosurgery 67:864-873 (1987)). Unfortunately, these therapies have been met with only moderate success.
Local treatments, such as radiation therapy and surgery, offer a way of reducing the tumor mass in regions of the body that are accessible through surgical techniques or high doses of radiation therapy. However, more effective local therapies with fewer side effects are needed. Moreover, these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients. To combat the spread of tumor cells, systemic therapies are used.
One such systemic treatment is chemotherapy. Chemotherapy is the main treatment for disseminated, malignant cancers (Slapak, C. A. and Kufe, D. W., “Principles of Cancer Therapy,” in Harrison's Principles of Internal Medicine, Fauci, A. S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527). However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. Id. This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. Id at 533. Another drawback to the use of chemotherapeutic agents is their severe side effects. Id. at 532. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. Id. Clearly, new approaches are needed to enhance the efficiency with which a chemotherapeutic agent can kill malignant tumor cells, while at the same time avoiding systemic toxicity.
2. Challenges Presented by Central Nervous System Tumors
Another problem in cancer treatment is that certain types of cancer, e.g., gliomas, which are the most common primary malignancy arising in the human brain, defy the current modalities of treatment. Despite surgery, chemotherapy, and radiation therapy, glioblastoma multiforme, the most common of the gliomas, is almost universally fatal (Schoenberg, in Oncology of the Nervous System, M. D. Walker, ed., Boston, Mass., Martinus Nijhoff (1983); Levin et al., Chapter 46 in Cancer: Principles and Practice of Oncology, vol. 2, 3rd ed., De Vita et al., eds., Lippincott Press, Philadelphia (1989), pages 1557-1611).
Gliomas represent nearly 40% of all primary brain tumors, with glioblastoma multiforme constituting the most malignant form (Schoenberg, “The Epidemiology of Nervous System Tumors,” in Oncology of the Nervous System, Walker, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983)). The five year survival rate for persons with this high grade type of astrocytoma is less than 5 percent, given the current treatment modalities (surgery, radiation therapy and/or chemotherapy) (Mahaley et al., Neurosurgery 71: 826-836 (1989); Schoenberg, in Oncology of the Nervous System, Walker, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983); Kim et al., J. Neurosurg. 74:27-37 (1991), Daumas-Duport et al., Cancer 2:2152-2165 (1988)). After treatment with radiation therapy, glioblastomas usually recur locally. Hochberg, F. H., et al., Neurology 30:907-911 (1980). Neurologic dysfunction and death in an individual with glioblastoma are due to the local growth of the tumor. Systemic metastases are rare. Id. For this reason, regional cancer therapy methods, rather than systemic methods, may be especially suitable for the treatment of glioblastomas.
Moreover, glioblastomas are resistant to many chemotherapeutic agents, perhaps due to the proliferative characteristics of this tumor type. Many chemotherapeutic agents are cell-cycle-active, i.e., cytotoxic primarily to actively cycling cells (Slapak, C. A., and Kufe, D. W., “Principles of Cancer Therapy,” in Harrison's Principles of Internal Medicine, Fauci, A. S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527). Generally, chemotherapy is most effective for cancers with a small tumor burden where the growth fraction of the tumor is maximal. Id. The growth fraction for glioblastoma tumors is only 30%, with the remaining 70% of cells being in G0, a resting phase (cells in G0 may die or may re-enter the active cell cycle (Yoshii et al., J. Neurosurg. 65:659-663 (1986)). While the 30% of glioblastoma cells that are actively dividing contribute to the lethal progression of this tumor, the 70% that are quiescent are responsible for the resistance of these tumors to a number of chemotherapeutic agents that target actively proliferating cells.
Unfortunately, regional treatments, such as surgery and radiation therapy have also found limited success in the treatment of glioblastomas (Burger et al., J. Neurosurg. 58:159-169 (1983); Wowra et al., Acta Neurochir. (Wien) 99:104-108 (1989); Zamorano et al., Acta Neurochir. Suppl. (Wien) 46:90-93 (1989)). Surgical treatment for glioblastomas is hampered by the lack of distinct boundaries between the tumor and the surrounding parenchyma, and by the migration of tumor cells in the white matter tracts extending out from the primary site (Burger et al., J. Neurosurg. 58:159-169 (1983)), which preclude their complete removal. Radiation therapy, which targets rapidly proliferating cells, is limited by the low growth fraction in glioblastomas, and by the radiation sensitivity of adjacent normal tissue (Wowra et al., Acta Neurochir. (Wien) 99:104-108 (1989); Zamorano et al., Acta Neurochir. Suppl. (Wien) 46:90-93 (1989)). Thus, new approaches are especially needed to treat brain tumors.
B. Non-Traditional Approaches to Cancer Therapy Using Viruses
One non-traditional approach to cancer therapy employs mutated viruses to target neoplastic cells. See, Chung, R. Y. and Chiocca, E. A., Surg. Oncol. Clin. N. Am. 7:589-602 (1998)).
Proposed viral cancer therapies include two distinct approaches: (1) direct cell killing (oncolysis) by a mutagenized virus (Martuza et al., Science 252:854-856 (1991); Mineta et al., Nature Med 1:938-943 (1995); Boviatsis et al., Cancer Res. 54: 5745-5751 (1994); Kesari, et al., Lab. Invest. 73: 636-648 (1995); Chambers et al., Proc. Natl. Acad. Sci. USA 92: 1411-1415 (1995); Lorence, R. M. et al., J. Natl. Cancer. Inst. 86: 1228-1233 (1994); Bischoff, et al., Science 274: 373-376 (1996); Rodriguez et al., Cancer Res. 57: 2559-2563 (1997)); and (2) the use of viral vectors to deliver a transgene whose expression product activates a chemotherapeutic agent (Wei et al., Human Gene Therapy 5: 969-978 (1994); Chen and Waxman, Cancer Res. 55: 581-589 (1995); Moolten, Cancer Gene Ther. 1: 279-287 (1994); Fakhrai et al., Proc. Natl. Acad. Sci. USA 93: 2909-2914 (1996); Roth et al., Nature Med. 2:985-991 (1996); Moolten, Cancer Res. 46: 5276-5281 (1986); Chen et al., Proc. Natl. Acad. Sci. USA 91: 3054-3057 (1994)).
1. Viral Oncolysis
The genetic engineering of viruses for use as oncolytic agents has initially focused on the use of replication-incompetent viruses. This strategy was hoped to prevent damage to non-tumor cells by the viruses. A major limitation of this approach was that these replication-incompetent viruses required a helper virus to be able to integrate and/or replicate in a host cell. One example of the viral oncolysis approach, the use of replication-defective retroviruses for treating nervous system tumors, requires the implantation of a producer cell line to spread the virus. These retroviruses are limited in their effectiveness, because each replication-defective retrovirus particle can enter only a single cell and cannot productively infect others thereafter. Therefore, they cannot spread far from the producer cell, and are unable to completely penetrate a deep, multilayered tumor in vivo (Markert et al., Neurosurg. 77:590 (1992); Rarn et al., Nature Medicine 3:1354-1361 (1997)).
More recently, genetic engineering of oncolytic viruses has focused on the generation of replication-conditional viruses in an attempt to avoid systemic infection, while allowing the virus to spread to other tumor cells. Replication-conditional viruses are designed to preferentially replicate in actively dividing cells, such as tumor cells. Thus, these viruses should target tumor cells for oncolysis, and replicate in these cells so that the virus can spread to other tumor cells.
Some recent strategies for creating replication-conditional viral mutants as anticancer agents have employed mutations in selected adenoviral or herpes simplex virus type 1 (HSV-1) genes to render them replication-conditional (Martuza, R. L., et al., Science 252:854-856 (1991); Mineta, T., et al., Nature Med 1:938-943 (1995); Boviatsis, E. J., et al., Cancer Res. 54: 5745-5751 (1994); Kesari, S., et al., Lab. Invest. 73: 636-648 (1995); Chambers, R., et al., Proc. Natl. Acad. Sci. USA 92: 1411-1415 (1995); Lorence, R. M. et al., J. Nat. Cancer. Inst. 86: 1228-1233 (1994); Bischoff, J. R., et al., Science 274: 373-376 (1996); Rodriguez, R., et al., Cancer Res. 57: 2559-2563 (1997)). For example, an adenovirus with a deletion in the E1B-55 Kd encoding gene has been shown to selectively replicate in p53-defective tumor cells (Bischoff, J. R., et al., supra).
Two broad types of replication-conditional HSV mutants in a single gene have been studied to date. The first consists of viral mutants with defects in the function of a viral gene needed for nucleic acid metabolism, such as thymidine kinase (Martuza, R. L., et al., Science 252:854-856 (1991)), ribonucleotide reductase (RR) (Goldstein, D. J. & Weller, S. K., J. Virol. 62:196-205 (1988); Boviatsis, E. J., et al., Gene Ther. 1:323-331 (1994); Boviatsis, E. J., et al., Cancer Res. 54:5745-5751 (1994); Mineta, T., et al., Cancer Res. 54:3363-3366 (1994)), or uracil-N-glycosylase (Pyles, R. B. and Thompson, R. I., J. Virol. 68:4963-4972 (1994)).
The second consists of viral mutants with defects in the function of the γ34.5 gene (Chambers, R., et al., Proc. Natl. Acad. Sci. USA 92:1411-1415 (1995)), which functions as a virulence factor by markedly enhancing the viral burst size of infected cells through suppression of the shutoff of host protein synthesis (Chou, J., et al., Science 250:1262-1266 (1990); Chou, J. and Roizman, B., Proc. Natl. Acad. Sci. USA 89:3266-3270 (1992)).
These single mutant strains have certain inherent limitations, including resistance to ganciclovir and acyclovir for TK mutants (Mineta, T., et al., Cancer Res. 54:3363-3366 (1994)), the risk of reversion to wild-type by a single recombination event with wild-type virus, and reduced oncolytic efficacy for γ34.5 mutants, at least in certain tumor cell lines (Kramm, C. M., et al., Hum. Gene Ther. 8:2057-2068 (1997); Mohr. 1. & Bluzman, Y., EMBO J. 15:4759-4766 (1996); Toda, M., et al., Hum. Gene Ther. 9:2177-2185 (1998)).
In an effort to decrease the risk of wild-type recombination, HSV viruses that are multiply mutated have been developed. These include mutants G207 (Mineta, T., et al., Nat. Med 1:938-943 (1995); U.S. Pat. No. 5,585,096, issued Dec. 17, 1996 to Martuza et al.), and MGH1 (Kramm, C. M., et al., Hum. Gene Ther. 8:2057-2068 (1997), which possess deletions of both copies of γ34.5 and an insertional mutation of RR.
Another multiply mutated HSV virus is the γ34.5/uracil DNA glycosylase (FNG) mutant strain 3616UB (Pyles, R. B., et al., Hum. Gene Ther. 8:533-544 (1997)). These double mutant strains demonstrate markedly reduced neurovirulence upon direct intracranial injection, retain sensitivity to ganciclovir, and show relatively selective replication in tumor cells compared to normal tissues. Such double mutant HSV strains retain the defective γ34.5 gene, thus demonstrating little virulence towards normal tissues. Although, they clearly demonstrate oncolytic effects against tumor cells, such effects are less than those observed in mutants with intact γ34.5 genes (Kramm, C. M., et al., Hum. Gene Ther. 8:2057-2068 (1997); Qureshi, N. and Chiocca, E. A., unpublished data). On the other hand, the toxicity exhibited by an intact γ34.5 gene might reduce the potential application of the latter viruses as oncolytic agents.
2. Viral Delivery of Anticancer Transgenes
The second approach in viral cancer therapy is the viral delivery of anticancer transgenes, whereby the phenotype of the target tumor cells is genetically altered to increase the tumor's drug sensitivity and responsiveness. This approach involves directly transferring a “chemosensitization” or “suicide” gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, F. L., Cancer Gene Therapy 1:279-287 (1994); Freeman, S. M., et al., Semin. Oncol. 23:31-45 (1996); Deonarain, M. P., et al., Gene Therapy 2: 235-244 (1995)).
Several prodrug activation genes have been studied for application in cancer gene therapy. In one example, herpes simplex virus thymidine kinase (HSV-TK) in combination with the prodrug ganciclovir represents a prototypic prodrug/enzyme activation system known in the art with respect to its potential applications in cancer gene therapy. HSV-TK phosphorylates the prodrug ganciclovir and generates nucleoside analogs that induce DNA chain termination and cell death in actively dividing cells. Tumor cells transduced with HSV-TK acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten, F. L. and Wells, J. M., J. Natl. Cancer Inst. 82:297-300 (1990); Ezzeddine, Z. D., et al., New Biol. 3:608-614 (1991).
In a second example, the bacterial gene cytosine deaminase (CD) is a prodrug/enzyme activation system that has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent (Mullen, C. A., et al., Proc. Natl. Acad. Sci. USA 89: 33-37 (1992); Huber, B. E., et al., Cancer Res. 53:4619-4626 (1993); Mullen, C. A., et al., Cancer Res. 54:1503-1506 (1994)).
Recent studies using these drug susceptibility genes have yielded promising results. See, e.g., Caruso, M., et al., Proc. Nat. Acad. Sci. USA 90:7024-7028 (1993); Oldfield, E., et al., Hum. Gene Ther. 4: 39 (1993); Culver, K, Clin. Chem 40: 510 (1994); O'Malley, Jr., B. W., et al., Cancer Res. 56:1737-1741 (1996); Rainov, N. G., et al., Cancer Gene Therapy 3:99-106 (1996).
Several other prodrug-activating enzyme systems have also been investigated (T. A. Connors, Gene Ther. 2:702-709 (1995)). These include the bacterial enzyme carboxypeptidase G2, which does not have a mammalian homolog, and can be used to activate certain synthetic mustard prodrugs by cleavage of a glutamic acid moiety to release an active, cytotoxic mustard metabolite (Marais, R., et al., Cancer Res. 56: 4735-4742 (1996)), and E. coli nitro reductase, which activates the prodrug CB 1954 and related mustard prodrug analogs (Drabek, D., et al., Gene Ther. 4:93-100 (1997); Green, N. K., et al., Cancer Gene Ther. 4:229-238 (1997)), some of which may be superior to CB11954 (Friedlos, F. et al., J Med Chem 40:1270-1275 (1997)). The principle underlying these approaches to prodrug activation gene therapy is that transduction of a tumor cell population with the foreign gene confers upon it a unique prodrug activation capacity, and hence a chemosensitivity which is absent from host cells that do not express the gene.
More recently, a drug activation/gene therapy strategy has been developed based on a cytochrome P450 gene (“CYP” or “P450”) in combination with a cancer chemotherapeutic agent that is activated through a P450-catalyzed monoxygenase reaction (Chen, L. and Waxman, D. J., Cancer Research 55:581-589 (1995); Wei, M. X., el al., Hum. Gene Ther. 5:969-978 (1994); U.S. Pat. No. 5,688,773, issued Nov. 18, 1997). Unlike the prodrug activation strategies mentioned above, the P450-based drug activation strategy utilizes a mammalian drug activation gene (rather than a bacterially or virally derived gene), and also utilizes established chemotherapeutic drugs widely used in cancer therapy.
Many anti-cancer drugs are known to be oxygenated by cytochrome P450 enzymes to yield metabolites that are cytotoxic or cytostatic toward tumor cells. These include several commonly used cancer chemotherapeutic drugs, such as cyclophosphamide (CPA), its isomer ifosfamide (IFA), dacarbazine, procarbazine, thio-TEPA, etoposide, 2-aminoanthracene, 4-ipomeanol, and tamoxifen (LeBlanc, G. A. and Waxman, D. J., Drug Metab. Rev. 20:395-439 (1989); Ng, S. F. and Waxman D. J., Intl. J. Oncology 2:731-738 (1993); Goeptar, A. R., et al., Cancer Res. 54:2411-2418 (1994); van Maanen, J. M., et al., Cancer Res. 47:4658-4662 (1987); Dehal, S. S., et al., Cancer Res. 57:3402-3406 (1997); Rainov, N. G., et al., Human Gene Therapy 9:1261-1273 (1998)).
In one example of this approach, tumor cells were rendered highly sensitive to CPA or IFA by transduction of CYP2B1, which encodes a liver P450 enzyme that exhibits a high rate of CPA and IFA activation (Clarke, L. and Waxman, D. J., Cancer Res. 49:2344-2350 (1989); Weber, G. F. and Waxman, D. J., Biochem. Pharmacol. 45:1685-1694 (1993)). This enhanced chemosensitivity has been demonstrated both in vitro and in studies using a subcutaneous rodent solid tumor model and human breast tumor grown in nude mice in vivo, and is strikingly effective in spite of the presence of a substantial liver-associated capacity for drug activation in these animals (Chen, L., et al., Cancer Res. 55:581-589 (1995); Chen, L., et al., Cancer Res. 56:1331-1340 (1996)). This P450-based approach also shows significant utility for gene therapy applications in the treatment of brain tumors (Wei, M. X., et al., Human Gene Ther. 5:969-978 (1994); Manome, Y., et al., Gene Therapy 3:513-520 (1996); Chase, M., et al., Nature Biotechnol. 16:444-448 (1998)).
In addition to transgenes comprising prodrug-activating or “suicide” genes, other types of anticancer transgenes have also been studied, including, cytokine genes (to enhance immune defense against the tumor) (Blankenstein, T., et al., J. Exp. Med. 173:1047-1052 (1991); Colombo, M. P., et al., Cancer Metastasis Rev. 16:421-432 (1997); Colombo, M. P., et al., Immunol. Today 15:48-51 (1994)) as well as other tumor toxic genes, such as diptheria toxin (Coll-Fresno, P. M., et al., Oncogene 14:243-247 (1997)), pseudomonas toxin, anti-angiogenesis genes, tumor vaccination genes, tumor suppressor genes, radiosensitivity genes, antisense RNA, and ribozymes (Zaia, J. A., et al., Ann. N.Y. Acad. Sci. 660:95-106 (1992)).
While both the virus-based and the gene-based approaches have provided evidence of significant therapeutic effects in animal models of tumors, each method suffers from inherent limitations. Although the viral-based approach theoretically provides the potential for extensive replication of the virus with spread in the tumor mass, its effects are limited by the efficiency of viral infection; the requirement of a helper virus or producer cell line for some viral vectors; tumor cell heterogeneity (Sidranski et al., 355: 846-847 (1992); Bigner et al., J. Neuropathol. Exp. Neurol. 40: 201-229 (1981)) for the cellular factor(s) complementing viral mutant growth for other viral vectors; and antiviral immune responses.
In the gene-based approaches tested thus far, the efficiency of transduction of cells within a tumor mass is limited by the defective nature of the vector. In fact, the majority of positively transduced cells occurs within a few cell layers from the site of vector inoculation (Nilaver et al. Proc. Natl. Acad. Sci. USA 21: 9829-9833 (1995); Muldoon et al., Am. J. Pathol. 147: 1840-1851 (1995); Ram Z. et al., J. Neurosurg. 82, 343A (abst.)(1995)). Moreover, even for viral vector systems where a producer cell line is unnecessary, or not killed by the suicide gene/drug combination, viral replication may be inhibited by the drug used. Furthermore, where the suicide-gene/drug combination is TK/GCV, the ability of the drug to kill tumor cells is limited by the stage of the cell cycle of the cells as GCV targets only cells in the process of DNA replication. It is thus unlikely that therapeutic gene delivery by these replication-defective vectors will affect tumor cells distant from the inoculation site, even in instances where the therapeutic gene produces a freely diffusible anticancer agent, such as cytokines or CPA metabolites.
A need therefore continues to exist for a safe and efficacious viral mutant that would provide a means to achieve selective virulence for tumors or other targeted cell populations, while retaining lack of toxicity for normal tissues.